Comparison of different techniques for detection of Gal-GalNAc, an early marker of colonic neoplasia

The tumor marker, D-galactose-B[1-31-Nacetyl- D-galactosamine (Gal-GalNAc, also known as Tantigen) can be identified by a very simple galactose oxidase-Schiff 's (GOS) reaction either on tissues or on rectal mucus samples from patients with colorectal neoplasms. Gal-GalNAc is expressed in the neoplastic mucosa as well as the remote non-neoplastic mucosa. It is, however, not expressed in colonic mucosa of normal subjects. We studied the expression of Gal-GalNAc by GOS reaction, lectin reactivity and immunocytochemistry in 10 normal, 45 precancerous [5 Crohn's disease, 15 ulcerative colitis (5 without dysplasia and 10 with dysplasia), 25 tubular adenomas], and 25 adenocarcinoma cases. Normal mucosa remote from tubular adenoma and adenocarcinoma was also studied. The GOS method was compared with reactivity of the lectin jacalin and immunostaining with antibody to T antigen (Anti-Tag Ab). GOS reaction was negative in all of the 10 normal specimens. Of the 5 Crohn's disease specimens, 2 were positive and 3 negative. In the 5 ulcerative colitis cases without dysplasia, positive reaction was seen in 2 cases and negative in 3. Of the 10 cases of ulcerative colitis with dysplasia, 5 showed positivity in dysplastic areas, and 3 of these were also positive in remote non dysplastic mucosa. Twenty of 25 tubular adenomas yielded a positive reaction in the adenoma, 14 of them showing positivity also in remote mucosa; 3 cases showed a positive reaction only in remote mucosa. Of the 25 adenocarcinomas, 21 showed a positive reaction in the adenocarcinoma as well as the remote mucosa. GOS reaction was intense in well differentiated adenocarcinoma and weak in poorly differentiated adenocarcinoma. Intense reaction was also seen in the intracellular mucus of some aberrant crypts and morphologically normal crypts remote from adenocarcinoma and tubular adenoma. GOS reaction showed an overall sensitivity of 75.7% and specificity of 100% for cancer and precancerous lesions. Jacalin reactivity was slightly more sensitive (84.3%) but less specific (80%) and Tag Ab reactivity even less sensitive (50%) but as specific (100%) for neoplastic and dysplastic mucosa. We conclude that the detection of the carbohydrate moiety Gal-GalNAc varies with the technique used. Compared to other techniques, GOS reaction is extremely simple and has a high degree of sensitivity and specificity. It can be used for detection of this tumor marker in remote non-neoplastic mucosa of patients with neoplasia or at risk of developing neoplasia. It, therefore, could be used as a cost effective screening test in rectal biopsy specimens of such patients

Comparison of different techniques for detection of Gal-GalNAc, an early marker of colonic neoplasia

The tumor marker, D-galactose-B[1-31-Nacetyl- D-galactosamine (Gal-GalNAc, also known as Tantigen) can be identified by a very simple galactose oxidase-Schiff 's (GOS) reaction either on tissues or on rectal mucus samples from patients with colorectal neoplasms. Gal-GalNAc is expressed in the neoplastic mucosa as well as the remote non-neoplastic mucosa. It is, however, not expressed in colonic mucosa of normal subjects. We studied the expression of Gal-GalNAc by GOS reaction, lectin reactivity and immunocytochemistry in 10 normal, 45 precancerous [5 Crohn's disease, 15 ulcerative colitis (5 without dysplasia and 10 with dysplasia), 25 tubular adenomas], and 25 adenocarcinoma cases. Normal mucosa remote from tubular adenoma and adenocarcinoma was also studied. The GOS method was compared with reactivity of the lectin jacalin and immunostaining with antibody to T antigen (Anti-Tag Ab). GOS reaction was negative in all of the 10 normal specimens. Of the 5 Crohn's disease specimens, 2 were positive and 3 negative. In the 5 ulcerative colitis cases without dysplasia, positive reaction was seen in 2 cases and negative in 3. Of the 10 cases of ulcerative colitis with dysplasia, 5 showed positivity in dysplastic areas, and 3 of these were also positive in remote non dysplastic mucosa. Twenty of 25 tubular adenomas yielded a positive reaction in the adenoma, 14 of them showing positivity also in remote mucosa; 3 cases showed a positive reaction only in remote mucosa. Of the 25 adenocarcinomas, 21 showed a positive reaction in the adenocarcinoma as well as the remote mucosa. GOS reaction was intense in well differentiated adenocarcinoma and weak in poorly differentiated adenocarcinoma. Intense reaction was also seen in the intracellular mucus of some aberrant crypts and morphologically normal crypts remote from adenocarcinoma and tubular adenoma. GOS reaction showed an overall sensitivity of 75.7% and specificity of 100% for cancer and precancerous lesions. Jacalin reactivity was slightly more sensitive (84.3%) but less specific Offprint requests to: Dr. A.M. Shamsuddin, Department of Pathology ,l0 S Pine Street, Baltimore, MD, USA 21201-1192. Fax: 410-706-8414 (80%) and Tag Ab reactivity even less sensitive (50%) but as specific (100%) for neoplastic and dysplastic mucosa. We conclude that the detection of the carbohydrate moiety Gal-GalNAc varies with the technique used. Compared to other techniques, GOS reaction is extremely simple and has a high degree of sensitivity and specificity. It can be used for detection of this tumor marker in remote non-neoplastic mucosa of patients with neoplasia or at risk of developing neoplasia. It, therefore, could be used as a cost effective screening test in rectal biopsy specimens of such patients

Production of î±-amylase using new strain of bacillus polymyxa isolated from sweet potato

In this study, a new amylase producer strain was isolated from sweet potato tuber. This strain was able to grow at 37 °C and produce a-amylase in high quantity compared to other standard strain cultures. In the first part, cultivation in shake flask in standard medium was carried out to give complete information about the growth and production kinetics of this strain. The results clearly demonstrate that the isolated strain is able to production a-amylase in submerged culture with concentration up to 2050 ?kat/L after 20 h cultivation. Furthermore, medium optimization was carried out by changing the starch concentration and cell cultivation in medium of mixed carbon source (composed of starch and glucose of ratio 15:5 g/g) to enhance the production process and to increase the growth rate. The volumetric and specific a-amylase production in this optimized medium were 4550 ?kat/L and 1060 ?kat/g, respectively. Further improvement in enzyme production process was achieved by scaling up the process from shake flask to 3-L stirred tank bioreactor under non-oxygen limiting condition. The maximal volumetric and specific a-amylase productions in bioreactor batch culture were 5210 ?kat/L and 1095?kat/g, respectively, after only 14 h cultivation

Ex Vivo and In Vivo Imaging and Biodistribution of Aptamers Targeting the Human Matrix MetalloProtease-9 in Melanomas

International audienceThe human Matrix MetalloProtease-9 (hMMP-9) is overexpressed in tumors where it promotes the release of cancer cells thus contributing to tumor metastasis. We raised aptamers against hMMP-9, which constitutes a validated marker of malignant tumors, in order to design probes for imaging tumors in human beings. A chemically modified RNA aptamer (F3B), fully resistant to nucleases was previously described. This compound was subsequently used for the preparation of F3B-Cy5, F3B-S-acetylmercaptoacetyltriglycine (MAG) and F3B-DOTA. The binding properties of these derivatives were determined by surface plasmon resonance and electrophoretic mobility shift assay. Optical fluorescence imaging confirmed the binding to hMMP-9 in A375 melanoma bearing mice. Quantitative biodistribution studies were performed at 30 min, 1h and 2 h post injection of 99mTc-MAG-aptamer and 111In-DOTA-F3B. 99mTc radiolabeled aptamer specifically detected hMMP-9 in A375 melanoma tumors but accumulation in digestive tract was very high. Following i.v. injection of 111In-DOTA-F3B, high level of radioactivity was observed in kidneys and bladder but digestive tract uptake was very limited. Tumor uptake was significantly (student t test, p<0.05) higher for 111In-DOTA-F3B with 2.0%ID/g than for the 111In-DOTA-control oligonucleotide (0.7%ID/g) with tumor to muscle ratio of 4.0. Such difference in tumor accumulation has been confirmed by ex vivo scintigraphic images performed at 1h post injection and by autoradiography, which revealed the overexpression of hMMP-9 in sections of human melanomas. These results demonstrate that F3B aptamer is of interest for detecting hMMP-9 in melanoma tumor

Ex Vivo and In Vivo Imaging and Biodistribution of Aptamers Targeting the Human Matrix MetalloProtease-9 in Melanomas.

The human Matrix MetalloProtease-9 (hMMP-9) is overexpressed in tumors where it promotes the release of cancer cells thus contributing to tumor metastasis. We raised aptamers against hMMP-9, which constitutes a validated marker of malignant tumors, in order to design probes for imaging tumors in human beings. A chemically modified RNA aptamer (F3B), fully resistant to nucleases was previously described. This compound was subsequently used for the preparation of F3B-Cy5, F3B-S-acetylmercaptoacetyltriglycine (MAG) and F3B-DOTA. The binding properties of these derivatives were determined by surface plasmon resonance and electrophoretic mobility shift assay. Optical fluorescence imaging confirmed the binding to hMMP-9 in A375 melanoma bearing mice. Quantitative biodistribution studies were performed at 30 min, 1h and 2 h post injection of 99mTc-MAG-aptamer and 111In-DOTA-F3B. 99mTc radiolabeled aptamer specifically detected hMMP-9 in A375 melanoma tumors but accumulation in digestive tract was very high. Following i.v. injection of 111In-DOTA-F3B, high level of radioactivity was observed in kidneys and bladder but digestive tract uptake was very limited. Tumor uptake was significantly (student t test, p<0.05) higher for 111In-DOTA-F3B with 2.0%ID/g than for the 111In-DOTA-control oligonucleotide (0.7%ID/g) with tumor to muscle ratio of 4.0. Such difference in tumor accumulation has been confirmed by ex vivo scintigraphic images performed at 1h post injection and by autoradiography, which revealed the overexpression of hMMP-9 in sections of human melanomas. These results demonstrate that F3B aptamer is of interest for detecting hMMP-9 in melanoma tumor

Ex vivo scintigraphic planar image of posterior paw at 1h after i.v. injection of ¹¹¹In-DOTA-F3B-aptamer and 111In-DOTA-control-aptamer.

<p>Ex vivo scintigraphic planar image of posterior paw at 1h after i.v. injection of ¹¹¹In-DOTA-F3B-aptamer and 111In-DOTA-control-aptamer.</p

Comparison of the results obtained by radiolabeling of representative tumor tissue sections with ¹¹¹In-DOTA-F3B aptamer (left image) and ¹¹¹In-DOTA-control sequence (right image).

<p>The difference of activity seems to increase in a tumor grade-dependent manner. (A) Lentigo malignant melanoma, (B) Nodular melanoma, (C) Mostly metastatic node.</p

Quantitative biodistribution of ^99mTc-MAG-aptamer and ^99mTc-MAG-control aptamer as function of post i.v. injection delay expressed as % of injected dose per gram of tissue.

<p>Quantitative biodistribution of ^99mTc-MAG-aptamer and ^99mTc-MAG-control aptamer as function of post i.v. injection delay expressed as % of injected dose per gram of tissue.</p

Quantitative biodistribution of ¹¹¹In-DOTA-F3B-aptamer and ¹¹¹In-DOTA-control-aptamer as function of post i.v. injection delay expressed as % of injected dose per gram of tissue.

<p>Quantitative biodistribution of ¹¹¹In-DOTA-F3B-aptamer and ¹¹¹In-DOTA-control-aptamer as function of post i.v. injection delay expressed as % of injected dose per gram of tissue.</p